Absorption meters have long been used to measure the light absorption of materials. Prior absorption meters are sophisticated electronic devices that are sensitive to transportation and environmental perturbations. As a result, such meters are not capable of performing in situ analyses of liquids at remote sites. To analyze liquids in situ such as naturally occurring bodies of water, an absorption meter must be (1) transportable, (2) protected from liquid immersion, (3) protected from the extreme pressures that occur with increased immersion depth, (4) operational over wide temperature variations, and (5) fortified for on-board deployment. Prior absorption meters are not capable of withstanding the effects of immersion in natural bodies of water.
Furthermore, natural bodies of water are analyzed by collecting and transporting samples to a location more convenient for analyzing the sample. Transporting samples may result in their contamination or alteration. For instance, liquid samples are often concentrated on filters. Filtration techniques introduce errors not only through the process of sample handling and filtration, but also due to the optical effects of discreetness.
A second example of perturbations associated with removing samples to a remote site involves the collection of phytoplankton for chlorophyll concentration determinations. Phytoplankton samples are collected at various ocean depths in sample vials that are then brought to the ocean surface. Such phytoplankton experience rapid depressurization and exposure to light. Both depressurization and light exposure affect phytoplankton physiology, which can affect the amount of chlorophyll detected from such phytoplankton. For instance, exposing chlorophyll a to fluorescent light can degrade the chlorophyll a to pheophytin in a matter of minutes. Pheophytin has an absorption that is roughly three times lower than that of chlorophyll a. Hence, collecting and transporting phytoplankton samples alters the amount of chlorophyll that is measured at the remote site compared to the true amount that would be measured in situ.
Moreover, analyzing samples at a remote location does not provide an accurate depiction of a dynamic liquid in real time. For biological inquiries, real time monitoring of a dynamic system is particularly important.
At present, the only viable instrument for in situ chlorophyll concentration measurements is the fluorometer. For instance, Soviet Union Patent No. 842511 describes a submersible fluorometer useful for measuring chlorophyll concentrations. Fluorescent measurements of chlorophyll are less reliable than absorption measurements mainly because fluorescence is a two-step process: (1) chlorophyll receives light energy at a first wavelength; and (2) chlorophyll emits light energy at a second wavelength. As a result, fluorescence depends on the quanta absorption rate per chlorophyll a concentration and the quanta emission per quanta absorption rate. Both of these factors vary as a function of light and nutrient conditions, and fluorescence efficiency may therefore vary as much as a factor of ten.
Fluorometers also have a large sampling time constant that is on the order of about three seconds, which means that a fluorometer must sample for about three seconds in order to achieve adequate resolution. At short time constants the resolution of fluorometers is only about 0.1 .mu.g/l. Also, fluorometers typically measure very low light levels, and use a flash lamp that has a large pulse-to-pulse instability. Hence, to get a precise measurement, a fluorometer must average over many-flash pulses. These two factors result in a large time sampling constant for fluorometers.
Moreover, phytoplankton with a set amount of chlorophyll will absorb a certain amount of light. However, the same phytoplankton's fluorescence depends on its physiological state, and the nutrient level.
Absorption has been used previously to determine the concentration of a wide variety of substances in a liquid. This method comprises preparing-known concentrations of a subject material and thereafter measuring the absorption of the material at a particular wavelength. This method is highly dependant upon obtaining accurate absorption data. For laboratory settings, external factors such as salinity and temperature can be stringently controlled. However, for in situ measurements, and especially for depth profile measurements of liquid bodies such as lakes and oceans, external conditions can not be as stringently controlled.
Chlorophyll concentration has been measured in the laboratory by determining the absorption of a liquid sample at about 675 nanometers (chlorophyll a has a strong absorbance band at approximately 675 nanometers) and at about 750 nanometers. The absorption at 750 nanometers is then subtracted from the absorption value at 675 nanometers to provide an absorption for chlorophyll a and pheophytin. We recently determined that the absorption coefficient is highly temperature dependent at about 750 nanometers. Hence, prior techniques that subtract the 750-nanometer absorption to determine the concentration of chlorophyll a introduce significant temperature-dependant absorption errors. Because chlorophyll a concentration measurements have typically been done at remote laboratory settings, it was not heretofore appreciated that in situ measurements (wherein the temperature is in constant flux), if an in situ absorbance meter had been available, would provide inaccurate chlorophyll a concentration data.
Light scattering is another problem encountered with differential absorption techniques for determining concentrations of particulate and dissolved substances. This problem has not been satisfactorily addressed by prior methods and devices used for concentrations involving differential absorption techniques. Light scattering can introduce significant error into absorption values, which in turn introduces error into the concentration values determined from this absorption data.